High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity

  • Rowlands M
  • Newbatt Y
  • Prodromou C
 et al. 
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The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8

Author-supplied keywords

  • Adenosinetriphosphatase
  • Colorimetry
  • Drug Screening Assays,Antitumor
  • Enzyme Inhibitors
  • Fungal Proteins
  • Heat-Shock Proteins
  • Heat-Shock Proteins 90
  • Phosphates
  • Proteins
  • Research
  • Research Support,Non-U.S.Gov't
  • Therapeutics
  • analysis
  • antagonists & inhibitors
  • isolation & purification
  • metabolism
  • methods
  • pharmacology

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  • PMID: 15051534


  • M G Rowlands

  • Y M Newbatt

  • C Prodromou

  • L H Pearl

  • P Workman

  • W Aherne

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